Heart-type fatty acid binding protein is a novel prognostic marker in patients with non-ischaemic dilated cardiomyopathy.

OBJECTIVE: To determine whether concentrations of heart-type fatty acid binding protein (H-FABP) measured before hospital discharge predict critical cardiac events in patients with idiopathic dilated cardiomyopathy (DCM). PATIENTS: 92 consecutive patients with DCM were enrolled and followed up for four years. MAIN OUTCOME MEASURES: Serum concentrations of H-FABP, brain natriuretic peptide (BNP), cardiac troponin T before hospital discharge and survival rate. RESULTS: 23 patients died of cardiac causes, received a left ventricular assist device or underwent heart transplantation during the four-year follow up. Univariate analyses showed that New York Heart Association functional class, heart rate, ejection fraction, serum H-FABP and plasma BNP were significant variables. According to multivariate analysis, serum H-FABP and plasma BNP concentrations were independent predictors of critical cardiac events. Cardiac troponin T before hospital discharge was not a predictor. The area under the receiver operating characteristic curve for death from critical cardiac events was similar between H-FABP and BNP. Patients with an H-FABP concentration at or above the median (> or = 5.4 ng/ml) had a significantly lower survival rate than those below the median, according to analysis by log rank test (p < 0.0001). When combined with BNP concentration at or above the median (> or = 138 pg/ml), H-FABP below the median predicted the worst prognosis among the combinations. CONCLUSIONS: The concentration of serum H-FABP before discharge from hospital may be an independent predictor for critical cardiac events in DCM.

Development of a simple whole blood panel test for detection of human heart-type fatty acid-binding protein.

OBJECTIVE: For the diagnosis of acute myocardial infarction (AMI), we have developed a rapid and simple whole blood panel test for the detection of human heart-type fatty acid-binding protein (H-FABP) using one-step immunochromatography. METHODS AND RESULTS: We have developed a whole blood panel test for rapid detection of human H-FABP using a one-step immunochromatography technique. The result of this panel test was not affected by the other contents of the blood such as bilirubin, hemoglobin and others. Furthermore, no cross-reactivity of the antibodies was found with other cardiac markers or other tissue-type FABPs. The result of this panel test was similar to the diagnostic cut-off value, 6.2 ng of H-FABP per mL of serum which was evaluated by the enzyme-linked immunosorbent assay (ELISA). CONCLUSION: We have developed a simple one-step immunochromatography technique to detect H-FABP in whole blood sample. Further studies are required to identify the value of this point-of-care testing (POCT) as a diagnostic marker for AMI.

Human heart-type cytoplasmic fatty acid-binding protein (H-FABP) for the diagnosis of acute myocardial infarction. Clinical evaluation of H-FABP in comparison with myoglobin and creatine kinase isoenzyme MB.

Heart-type fatty acid-binding protein (H-FABP) is a low molecular weight cytoplasmic protein and present abundantly in the myocardium. When the myocardium is injured, as in the case of myocardial infarction, low molecular weight cytoplasmic proteins including H-FABP are released into the circulation and H-FABP is detectable in a blood sample. We have already developed a direct sandwich-ELISA for quantification of human H-FABP using two distinct types of monoclonal antibodies specific for human H-FABP. In this study we investigated the clinical validity of H-FABP as a biochemical diagnostic marker in the early phase of acute myocardial infarction (AMI). To evaluate the diagnostic usefulness of H-FABP in the early phase of AMI, blood samples were obtained from the following patients within 12 hours after the appearance of symptoms, and serum levels of H-FABP were compared with those of conventional diagnostic markers, such as myoglobin and creatine kinase isoenzyme MB (CK-MB). Blood samples were collected from patients with confirmed AMI (n=140), patients with chest pain who were afterwards not classified as AMI by normal CK-MB levels (non-AMI) (n=49) and normal healthy volunteers (n=75). The serum concentration of H-FABP was quantified with our direct sandwich-ELISA. The concentration of myoglobin mass was measured with a commercial RIA kit. The serum CK-MB activity was determined with an immuno-inhibition assay kit. The overall sensitivity of H-FABP, within 12 hours after the appearance of symptoms, was 92.9%, while it was 88.6% with myoglobin and 18.6% with CK-MB. The overall specificity of H-FABP was 67.3%, while it was 57.1% with myoglobin and 98.0% with CK-MB. The diagnostic efficacy rates with these markers were 86.2% (H-FABP), 80.4% (myoglobin) and 39.2% (CK-MB), respectively. The diagnostic validity of H-FABP was further assessed by receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) of H-FABP was 0.921, which was significantly greater than with myoglobin (AUC: 0.843) and CK-MB (AUC: 0.654). These parameters, such as sensitivity, specificity, diagnostic efficacy and diagnostic accuracy, obtained for patients with chest pain within 3 hours and/or 6 hours after the onset of symptoms were almost the same as those for patients within 12 hours after symptoms. H-FABP is more sensitive than both myoglobin and CK-MB, more specific than myoglobin for detecting AMI within 12 hours after the onset of symptoms, and shows the highest values for both diagnostic efficacy and ROC curve analysis. Thus, H-FABP has great potential as an excellent biochemical cardiac marker for the diagnosis of AMI in the early phase.

Development and evaluation of a direct sandwich-enzyme-linked immunosorbent assay for the quantification of human hepatic triglyceride lipase mass in human plasma.

We have developed a direct sandwich-enzyme-linked immunosorbent assay (ELISA) for quantification of the hepatic triglyceride lipase (HTGL) immunoreactive mass in human plasma. This direct sandwich-ELISA uses a combination of two distinct monoclonal antibodies (MAbs), which recognize different epitopes on the HTGL molecule: a horseradish peroxidase (HRP)-labeled anti-human HTGL MAb (2(4)F12C12) as an enzyme-linked MAb, and an anti-human HTGL MAb (1(11)A3H3) coated on a microtiter plate as a solid-phase MAb. Purified human post-heparin plasma (PHP)-HTGL was used as the standard material. The detection range of the sandwich-ELISA was 40-800 ng of HTGL protein per ml of plasma. The intra- and inter-assay coefficients of variation were less than 2.0% and 2.3%, respectively. The recovery tests resulted in variation only between 97.7% and 103.5%. No significant assay interference was caused by a high concentration of triglyceride, hemoglobin, bilirubin, uric acid, or creatinine. The reliability of the HTGL mass values obtained with the direct sandwich-ELISA was assessed by comparison with the HTGL mass values determined by our earlier one-step sandwich-enzyme immunoassay (EIA). The two sets of values showed a highly significant correlation (r=+0.952, n=64). Strong correlation (r=+0. 959, n=50) was also found between the HTGL masses with the direct sandwich-ELISA and the HTGL activities determined with a selective immunoinactivation assay. The HTGL mass concentrations in PHP from 64 healthy subjects were 1916+/-841 ng/ml by the direct sandwich-ELISA and 1925+/-785 ng/ml (mean+/-standard deviation (SD)) by the one-step sandwich-EIA. The present direct sandwich-ELISA permits rapid identification of certain HTGL abnormalities in PHP samples from patients with hypertriglyceridemia or diseases such as hypothyroidism or renal failure, which affect HTGL.

Development and evaluation of a direct sandwich enzyme-linked immunosorbent assay for the quantification of lipoprotein lipase mass in human plasma.

OBJECTIVE: The purpose of this study was to develop and evaluate a direct sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of the lipoprotein lipase (LPL) immunoreactive mass in human plasma using monoclonal antibodies (MAbs) directed against LPL purified from human postheparin plasma (PHP) [corrected]. METHODS AND RESULTS: The direct sandwich-ELISA was performed using a combination of two distinct types of MAbs that recognize different epitopes on the LPL molecule. The immunoreactive mass of human LPL was specifically measured using a horseradish peroxidase-labeled anti-human LPL MAb [1(1)D2B2] as an enzyme-linked MAb, and an anti-human LPL MAb [2(10)F8F9] coated on a polystyrene microtiter plate as a solid-phase MAb. Purified human PHP-LPL was used as a standard material. The detection range of the sandwich-ELISA was 3.6-460 ng of LPL protein per mL of plasma. The intra- and interassay coefficients of variation were less than 5.9% and 3.3%, respectively. The validity of this method was additionally assured by the recovery test, which resulted in the variation only between 97.5% and 105.1%, and also by the interference test, which resulted in noninterference of LPL assay with a high concentration of triglyceride, hemoglobin, bilirubin, uric acid, or creatinine. To assess the reliability of the LPL mass values obtained with the direct sandwich-ELISA, they were compared with LPL mass values determined by the one-step sandwich-EIA (MARKIT-F LPL EIA kit) previously established by us. This comparison showed a highly significant correlation (r = +0.990) between the two sets of values. The LPL mass concentrations in PHP from 33 healthy subjects were 267 +/- 53 and 257 +/- 59 ng/mL (mean +/- SD), respectively. CONCLUSION: The present direct sandwich-ELISA is useful for rapidly identifying certain abnormalities of LPL in PHP samples from patients with hypertriglyceridemia [corrected].

[The earlier detection of myocardial damage in open heart surgery using serum human heart fatty acid-binding protein].

Human heart fatty acid-binding protein (HH-FABP), which is a low molecular weight protein and abundant in the cytoplasm of myocardial cells, is reported to be released into the circulation shortly after the onset of acute myocardial damage. However, the changes in serum HH-FABP levels in open heart surgery have not been elucidated. To determine whether HH-FABP enables the earlier detection of myocardial damage caused by ischemia-reperfusion in open heart surgery, we measured the serial levels of serum HH-FABP, CK-MB and Troponin T (TnT) at every 15 min for 48 hours after reperfusion in 10 adult patients with coronary artery bypass graft. The serum HH-FABP levels reached the peak within 60 min after reperfusion (mean +/- SD; 49 +/- 7 min), and this was significantly (p < 0.001) earlier than CK-MB (212 +/- 108 min) and TnT (244 +/- 150 min). The peak value of serum HH-FABP had a significant correlation to the peak value of serum CK-MB or TnT (r = 0.815, p = 0.02; r = 0.925, p = 0.0001, respectively). These results indicate that serum HH-FABP enables the earlier detection of myocardial damage than the other markers in the patients with open heart surgery.

Human heart-type cytoplasmic fatty acid-binding protein as an indicator of acute myocardial infarction.

Human heart-type cytoplasmic fatty acid-binding protein (HH-FABPc) has been proposed as an early biochemical indicator of acute myocardial infarction (AMI). However, skeletal muscles also contain HH-FABPc identical to that found in the heart. Before HH-FABPc can be clinically employed as an indicator of AMI, its content in various tissues other than the heart must be known. Accordingly, we measured the HH-FABPc content of various human muscles and organs, using a sandwich enzyme-linked immunosorbent assay (ELISA) for HH-FABPc. HH-FABPc was abundant in the ventricles (0.46 mg/g wet weight and 1.5% of the cytoplasmic protein in the left ventricle), while the atria contained slightly less HH-FABPc (0.25 mg/g wet weight and 0.7% of the cytoplasmic protein in the left atrium). Of the skeletal muscles tested, the diaphragm contained about one-quarter of the HH-FABPc content of the heart, but other skeletal muscles contained very low levels of this protein. Other than the muscles, the kidneys contained less than one-tenth of the HH-FABPc in the heart, and negligible amounts were found in the liver and small intestine. The distribution of HH-FABPc in the heart and skeletal muscles was comparable to that of cardiac-specific creatine kinase (CK-MB) activity, and was inverse to the distribution of myoglobin. The plasma myoglobin/HH-FABPc ratio, determined in patients with AMI and those without AMI, closely reflected that in the heart and skeletal muscles. These findings indicate that HH-FABPc may be useful as a specific indicator of AMI, and the plasma myoglobin/HH-FABPc ratio could provide valuable information for the diagnosis of AMI.

Development of a sandwich enzyme-linked immunosorbent assay for the determination of human heart type fatty acid-binding protein in plasma and urine by using two different monoclonal antibodies specific for human heart fatty acid-binding protein.

We have developed a sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of human heart type fatty acid-binding protein (H-FABP) in human plasma and urine using the combination of two distinct monoclonal antibodies (MAbs) directed against human H-FABP purified from human heart muscle. The total assay time of the ELISA is practically much shorter than that of the competitive enzyme immunoassay (EIA) we previously reported. The immunoreactive mass of human H-FABP was specifically measured using a horseradish peroxidase (HRPO)-labeled anti-human H-FABP MAb as an enzyme-linked MAb, and anti-human H-FABP MAb immobilized on the polystyrene microtiter plate as a solid-phase MAb, and purified human H-FABP as standard materials. The assay range of the ELISA was 0-250 ng/ml of plasma and urine. The ELISA yielded a coefficient of variation of less than 10% in inter- and intra-assays, and the good linearity was obtained in dilution test using clinical samples. Anticoagulants, except sodium fluoride and a high concentration of hemoglobin and bilirubin, did not interfere with the assay of plasma samples. A high concentration of hemoglobin, bilirubin and immunoglobulin, and contamination with seminal plasma did not interfere with the assay of urine samples. The average recovery of purified human H-FABP added to human plasma and urine samples was 98.5% and 97.0%, respectively. Myoglobin and myosin did not crossreact in the ELISA. The minimum detection limit of the ELISA was 1.25 ng/ml. The immunoreactive masses of human H-FABP in plasma and urine samples, obtained from one hundred normal healthy subjects were quantified by the sandwich ELISA. The normal mean (+/- SD) level of human H-FABP mass in plasma was 3.65 +/- 1.81 ng/ml, and that in urine was 3.20 +/- 2.70 ng/ml. In conclusion, this sandwich ELISA is a useful tool for the sensitive and precise determination of human H-FABP in human plasma and urine, and it may be used specifically for clinical investigation and diagnosis of myocardial injury.

A sandwich-enzyme immunoassay for the quantification of lipoprotein lipase and hepatic triglyceride lipase in human postheparin plasma using monoclonal antibodies to the corresponding enzymes.

We have developed a sandwich-enzyme immunoassay (EIA) for the quantification of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) in human postheparin plasma (PHP) using monoclonal antibodies (MAbs) directed against the corresponding enzymes purified from human PHP. The sandwich-EIA for LPL was performed by using the combination of two distinct types of anti-LPL MAbs that recognize different epitopes on the LPL molecule. The immunoreactive mass of LPL was specifically measured using a beta-galactosidase-labeled anti-LPL MAb as an enzyme-linked MAb, an anti-LPL MAb linked with the bacterial cell wall as an insolubilized MAb, and purified human PHP-LPL as a standard. The sandwich-EIA for HTGL was carried out by using two distinct anti-HTGL MAbs that recognize different epitopes on HTGL. The limit of detection was 20 ng/ml for LPL and 60 ng/ml for HTGL. Each method yielded a coefficient of variation of less than 6% in intra- and inter-assays, and a high concentration of triglyceride did not interfere with the assays. The average recovery of purified human PHP-LPL and -HTGL added to human PHP samples was 98.8% and 97.5%, respectively. The immunoreactive masses of LPL and HTGL in PHP samples, obtained at a heparin dose of 30 IU/kg, from 34 normolipidemic and 20 hypertriglyceridemic subjects were quantified by the sandwich-EIA. To assess the reliability of the measured mass values, they were compared with the corresponding enzyme activities measured by selective immunoinactivation assay using rabbit anti-human PHP-LPL and -HTGL polyclonal antisera. Both assay methods yielded a highly significant correlation in either normolipidemic (r = 0.945 for LPL; r = 0.932 for HTGL) or hypertriglyceridemic subjects (r = 0.989 for LPL; r = 0.954 for HTGL). The normal mean (+/- SD) level of lipoprotein lipase mass and activity in postheparin plasma was 223 +/- 66 ng/ml and 10.1 +/- 2.9 mumol/h per ml, and that of hepatic triglyceride lipase mass and activity was 1456 +/- 469 ng/ml and 26.4 +/- 8.7 mumol/h per ml, respectively. The present sandwich-enzyme immunoassay methods make it possible to study the molecular nature of LPL and HTGL in PHP from patients with either primary or secondary hyperlipoproteinemia.

Simple enzyme immunoassay methods for recombinant human tumor necrosis factor alpha and its antibodies using a bacterial cell wall carrier.

We have developed simple methods for measuring recombinant human tumor necrosis factor alpha (rHu-TNF alpha) and antibodies to rHu-TNF alpha in the sera of animals intravenously injected with rHu-TNF alpha. rHu-TNF alpha was measured by a competitive binding enzyme immunoassay (C-EIA) using standard rHu-TNF alpha, beta-galactosidase labeled rHu-TNF alpha as enzyme-labeled antigen (E-Ag) and anti-rabbit IgG goat immunoglobulins coupled to bacterial cell walls (insolubilized second antibody). In contrast, anti-rHu-TNF alpha antibodies were measured by a sandwich EIA (S-EIA) using purified anti-rHu-TNF alpha rabbit IgG as standard, beta-galactosidase labeled rHu-TNF alpha as E-Ag, and rHu-TNF alpha coupled to bacterial cell walls as insolubilized antigen. C-EIA permits the determination of serum rHu-TNF alpha within the range of 2-150 U/ml (about 0.7-52 ng/ml) with a CV of below 7.6% and 99% recovery. S-EIA permits the determination of anti-rHu-TNF alpha antibodies within the range of 70-1000 ng/ml with a CV of less than 4% and 94.8-106.9% recovery.